Phytate is the major storage form of phosphorus in cereals and legumes. However, monogastric animals such as pig, poultry and fish are not able to metabolise or absorb phytate (or phytic acid) and therefore it is excreted, leading to potential phosphorous pollution in areas of intense livestock production. Moreover, phytic acid also acts as an antinutritional agent in monogastric animals by chelating metal agents such as calcium, copper and zinc.
In order to provide sufficient phosphates for growth and health of these animals, inorganic phosphate is added to their diets. Such addition can be costly and further increases potential pollution problems.
Through the action of phytase, phytate is generally hydrolysed to give lower inositol-phosphates and inorganic phosphate. Phytases are useful as additives to animal feeds where they improve the availability of organic phosphorus to the animal and decrease phosphate pollution of the environment (Wodzinski R J, Ullah A H. Adv Appl Microbiol. 42, 263-302 (1996)).
A number of phytases of fungal origin (Wyss M. et al. Appl. Environ. Microbiol. 65 (2), 367-373 (1999); Berka R. M. et al. Appl. Environ. Microbiol. 64 (II), 4423-4427 (1998); Lassen S. et al. Appl. Environ. Microbiol. 67 (10), 4701-4707 (2001)) and bacterial origin (Greiner R. et al. Arch. Biochem. Biophys. 303 (I), 107-113 (193); Kerovuo et al. Appl. Environ. Microbiol. 64 (6), 2079-2085 (1998); Kim H. W. et al. Biotechnol. Lett. 25, 1231-1234 (2003); Greiner R. et al. Arch. Biochem. Biophys. 341 (2), 201-206 (1997); Yoon S. J. et al. Enzyme and microbial technol. 18, 449-454 (1996); Zinin N. V. et al. FEMS Microbiol. Lett. 236, 283-290 (2004)) have been described in the literature. Specifically BP11 (WO2006/043178), BP17 (WO2008/097619) and BP111 (WO2009/129489) are known variant phytases suitable for use in food and animal feed due to their stability, especially their thermal stability. These phytases are variants of the wild-type phytase of Buttiauxella P1-29 (deposited under accession number NCIMB 4124). Phytase is known to undergo reversible thermal inactivation (Wyss, Appl. Envir. Microbiol. (1998) 64:4446).
Animal feeds may be made, produced and processed at high temperatures. In particular they may be formed by pellets or granules such as those described in WO99/32595 and WO2007/044968. Production of animal feeds therefore requires the feeds and feed ingredients to be thermostable at high temperatures. It is therefore advantageous if a feed enzyme, particularly phytase, retains a high level of enzymatic activity after exposure to high or elevated temperatures.
Enzymes may be modified at the amino acid level to introduce glycosylation sites, which promote the glycosylation of the enzyme. For example, an N-linked glycan is attached to the asparagine residue within the motif NXS/T on the surface of a secreted protein (Weerapana and Imperiali (2006) Glycobiology 16:91 R-101R). It is known that glycosylation of an enzyme can provide increased thermostability (Koseki et al. (2006) Biosci. Biotechnol. Biochem. 70: 2476-2480). It is also known that increased glycosylation can increase the protease stability at low pH of some phytases (WO01/90333) as well as thermostability (WO2006/028684). However, these references do not disclose phytase of Butiiauxella origin, or that further increased glycosylation improves resistance to the steam treatment during feed pelleting when the enzyme is in solid state.
The present teachings provide improved glycosylated phytases with increased inactivity reversibility. In some embodiments, the phytases can be used in food or feed. In some embodiments, the phytases undergo a pelleting process for inclusion in the food or feed.